Transient receptor potential V1 modulates neuroinflammation in Parkinson’s disease dementia: Molecular implications for electroacupuncture and rivastigmine

Objective(s): Parkinson’s disease (PD) is a common progressive neurodegeneration disease. Its incidence increases with age and affects about 1% of people over 60. Incidentally, transient receptor potential V1 (TRPV1) and its relation with neuroinflammation in mouse brain has been widely reported. Materials and Methods: We used 6-hydroxydopamine (6-OHDA) to induce PDD in mice. We then used the Morris water maze and Bio-Plex to test learning and inflammatory mediators in mouse plasma. Western blotting and immunostaining were used to examine TRPV1 pathway in the hippocampus and medial prefrontal cortex (mPFC). Results: On acquisition days 3 (Control = 4.40 ± 0.8 sec, PDD = 9.82 ± 1.52 sec, EA = 5.04 ± 0.58 sec, Riva = 4.75 ± 0.87 sec; P=0.001) and 4, reversal learning days 1, 2, 3 (Control = 2.86 ± 0.46 sec, PDD = 9.80 ± 1.83 sec, EA = 4.6 ± 0.82 sec, Riva = 4.6 ± 1.03 sec; P=0.001) and 4, PDD mice showed significantly longer escape latency than the other three groups. Results showed that several cytokines were up-regulated in PDD mice and reversed by EA and rivastigmine. TRPV1 and downstream molecules were up-regulated in PDD mice and further reversed by EA and rivastigmine. Interestingly, α7 nicotinic receptors and parvalbumin levels in both the hippocampus and prefrontal cortex increased in EA-treated mice, but not in rivastigmine-treated mice. Conclusion: Our results showed that TRPV1 played a role in the modulation of neuroinflammation of PDD, and could potentially be a new target for treatment.


Introduction
Parkinson's disease (PD) is the second most common neurodegenerative disease worldwide with increasing rates in elderly populations. Approximately 83% of patients with PD display dementia within 20 years of diagnosis (1). Even in its early stages , 26.7% exhibit mild cognitive impairments (2) which include working memory decline, cognitive inflexibility, and hallucinations (3). These problems impair the patients' quality of life and impose a significant burden on caregivers (4). Cognitive decline is associated with α-synuclein, tau, and amyloid pathologies and likely involves inflammation and different neurotransmitter systems (5). Because inflammatory responses are amplified by cytokines (IL-1β, TNF-α, IL-6, and IFN-γ) released into the blood via microglial activation (6), neuroinflammation is significantly related to cognitive decline (7).
Accumulating evidence suggests that the transient receptor potential vanilloid type 1 (TRPV1) channel is closely related to immune responses and might be considered a molecular switch for neuroinflammation in many neurodegenerative diseases including PD (8). This protein is a nonselective calcium-permeable cation channel that is highly sensitive to temperature and found in mammals adapted to harsh environments such as polar regions and deserts (9). It is activated by noxious heat, low pH, and animal toxins such as 6-hydroxydopamine (6-OHDA) (10). Brain TRPV1 can potentially detect harmful stimuli and plays a key role in microglia-to-neuron communication. It is highly expressed in microglial cells, which are responsible for inflammation (11) and expressed throughout the central nervous system (CNS), where it potentially supports atypical neurotransmission systems involved in multiple functions through the modulation of neuronal and glial activity (8).
A reduced incidence of PD in smokers has been recognized since the early 1960s (12). Populationbased studies show that smokers have an approximately 30%-50% reduced risk of developing PD (13), indicating the importance of nicotinic receptors. The involvement of nicotine receptors could explain the close anatomical relationship between nicotinic cholinergic TRPV1 in Parkinson's disease dementia Tsai et al. and dopaminergic neurotransmitter systems in the striatum (14). PD has been considered primarily as a dopaminergic disorder, but multiple CNS systems including cholinergic pathways, are currently thought to be involved in its pathogenesis (15). Several studies using functional imaging, such as proton emission tomography, demonstrate cortical cholinergic dysfunction in patients with PD and cognitive impairment (16). One pathologic investigation has found cholinergic neuronal loss in the nucleus basalis of Meynert in 11 patients with PD, but not in 13 age-matched control subjects (17). Furthermore, clinical trials (18) confirm the treatment efficacy of cholinesterase inhibitors (rivastigmine and donepezil) in patients with PD and cognitive impairment. Such improvement could decrease caregiver distress, including distress resulting from hallucinations (19).
Other studies show that α7-nicotinic acetylcholine receptors (α7-nAChRs) have strong links to inflammation and neurodegeneration (20), while others show that α7 nicotinic receptor agonists might decrease neuroinflammations (21). Acupuncture has been used for at least 3000 years to treat a variety of diseases (22), and complementary and alternative medicine (CAM) with acupuncture in real-world practice is a key component of treating PD worldwide (23). In fact, 63% of patients with PD in Korea (24), 50% in Argentina (25), 39% in Sweden (26), and 25% in Singapore (27) use at least one type of CAM, including acupuncture. More than 20 randomized controlled trials clinically support the efficacy of PD treatment with acupuncture (23). A review of basic studies (28)shows the following mechanisms of acupuncture: neuroprotection, cell proliferation, anti-apoptosis, anti-oxidant, and antiinflammation. Furthermore a recent study from South Korea demonstrates acupuncture-induced protection of dopaminergic neurons, regulation of gut microbiota, and inhibition of neuroinflammation in mice (29).
In this study, we have shown that neuroinflammatory mediators are up-regulated in PD dementia (PDD). More importantly, the results of our PDD mouse model have shown that TRPV1 and its related molecules play a role in the modulation of neuroinflammation. Because patients prefer either Western medicine or acupuncture, we have compared these treatment types, focusing on cognitive function. We have found that electroacupuncture (EA) and rivastigmine significantly reduced PDD via modulation of TRPV1 signaling. Our data recommend the use of EA and rivastigmine in treating PDD.

Experimental animals
We used a newborn subcutaneous 6-OHDA injection mouse model as previously described (30). Thirtysix newborn C57/BL6 mice were randomly assigned to four groups of nine individual animals. The four groups were: control (normal mice), PDD, EA (PDD + electroacupuncture), and Riva (PDD + oral rivastigmine). Mice in the latter three groups were anesthetized with 0.5% isoflurane and given subcutaneous injections of 6-OHDA (100 mg/kg dissolved in 0.1% ascorbic acid in 0.9% NaCl; Sigma, St Louis, Missouri, USA) in the middorsal region for four consecutive days soon after birth. Mice in the control group received vehicle (0.1% ascorbic acid in 0.9% NaCl). Animals were housed in Plexiglas cages with access to standard mouse chow and water ad libitum. Cages were located in a temperature-controlled room (23 °C-27 °C) under a 12:12 hr light-dark cycle (from 6:00 a.m. to 6:00 p.m.) with a relative humidity of 55%-65%. The experiment started at postnatal week eight. Mice weighed 16-23 g at this time. Experimental protocols were approved by the Institute of Animal Care and Use Committee of the China Medical University (Protocol number: CMUIACUC-2020-226), Taiwan, following the Guide for Use of Laboratory Animals (National Academies Press). We tried to minimize the number of animals used and their suffering.

Electroacupuncture
Mice in the EA group received electroacupuncture starting on week eight. Animals were treated six times, one time every other day, similar to real-world acupuncture treatment schedules. Stainless steel acupuncture needles (1.5 inch, 32G, Yu Kuang, Taiwan) were inserted bilaterally at KI3 to a depth of 1-2 mm. KI3 was located on the medial aspect of the foot, posterior to the medial malleolus and anterior to the tendon calcaneus (30). Square pulse (100 μs duration) electrical stimulation was delivered for 20 min at 2 Hz and 1 mA. Acupuncture treatments were administered between 11:00 to 14:00.

Oral rivastigmine
Mice in group four (Riva) were administered oral rivastigmine starting in week eight, once per day for 12 days. This schedule mimicked everyday use of oral rivastigmine in real-world practice. We used the human liquid formulation of rivastigmine, 120 ml/bottle, containing rivastigmine, 2 mg/ml, produced by Center Laboratories, Inc., Taiwan. We calculated the dose, dissolved the drug in 0.9% NaCl, and administered the solution by gavage.

Behavioral examination
A circular swimming pool (75 cm in diameter and 22 cm in height) was filled with water, 18 cm deep and maintained at 25 °C. Two principal axes of the maze were defined, with each line bisecting the maze perpendicular to the other to create an imaginary "+." Ends of each line demarcate the four cardinal directions: North (N), South (S), East (E), and West (W). South (S) was the experimenter's position, N is the opposite point. We put visual cues around the tank, with white square at the west location, circle in the north location, and triangle in the east location. Locations of visual cues were the same during 16 acquisition and 16 reversal trials for each mouse. A 7 x 7 cm transparent platform was placed 0.5 cm below the surface of the water in the defined area. Data were collected with a digital camera fixed at the top of the room and connected to a computer running Smart V.3 software (TrackMot V.5.45; Signa Technology Company, Taipei, Taiwan). This software measures mouse images to identify the center of its body and track its movement. We first acquired data to test spatial memory of mice. Each day of acquisition included four trials (31). We calculated mean values to generate Figure 1. After four days of acquisition, we changed the transparent platform to the opposite position of the tank to test reversal learning. The reversal learning involved four days, four trials per day. The starting locations of each trial are provided in Table 1. The interval between trials was about five min. Recording started when the camera detected the center of animal mass for two seconds. Recording would stop if the center of mass entered the transparent platform and remained for two seconds. Recording during each trial was 90 sec. If an animal did not reach the platform in time, the experimenter would guide it to the correct position and hold it in place for two seconds. After recording, we used the Smart software to calculate the escape latency, and swimming speed. Daily results are presented in Figure 1 as means and standard errors (SEM). After each trial, we used a heat lamp to warm mice to ensure maintenance of body temperature. The entire behavior test was performed by the same experimenter at the same time (11:00-14:00).

Bio-Plex ELISA
After behavior testing, mice were euthanized with 5% isoflurane by inhalation. Blood was collected from the orbital sinus into 3 ml BD Vacutainer glass tubes with 5.4 mg K2 EDTA and 2 ml BD Vacutainer glass tubes with 3 mg sodium fluoride and 6 mg Na2 EDTA. The samples were centrifuged at 1000 rpm/min for 10 min at 25 °C. Separated plasma was collected into 1.5 ml microcentrifuge tubes and stored at −80 °C. Plasma was analyzed using Bio-Plex cytokine assays (BIO-RAD, CA, USA). Four replicates were included.

Immunofluorescence
In each group, we randomly chose three mice to do the immunofluorescence. We euthanized with 5% isoflurane via inhalation, and intracardially perfused with normal saline followed by 4% paraformaldehyde. The brain was immediately dissected and post fixed with 4% paraformaldehyde at 4 °C for 3 days. The tissues were placed in 30% sucrose for cryoprotection overnight at 4 °C. The brain was embedded in optimal cutting temperature (OCT) compound and rapidly frozen using liquid nitrogen before storing the tissues at -80 °C. Frozen segments were cut at 20-um width on a cryostat then instantaneously placed on glass slides. The samples were fixed with 4% paraformaldehyde, and then incubated with blocking solution, consisting of 3% BSA, 0.1% Triton X-100, and 0.02% sodium azide, for 1 hr at room temperature. After blocking, the samples were incubated with primary antibody (1:200, Alomone, Jerusalem, Israel), TRPV1, prepared in 1% bovine serum albumin solution at 4 °C overnight. Afterward, the samples were incubated with the secondary antibody (1:500), 488-conjugated AffiniPure donkey anti-rabbit IgG (H +L), 594-conjugated AffiniPure donkey anti-goat IgG (H + L), and Peroxidase-conjugated AffiniPure donkey antimouse IgG (H + L) for 2 hr at room temperature before being fixed with cover slips for immunofluorescence visualization. The samples were observed by an epifluorescent microscope (Olympus, BX-51, Tokyo, Japan) with 20x numerical aperture (NA=0.4) objective. The images were analyzed by NIH ImageJ software (Bethesda, MD, USA).

Data analysis
The data of this study have been expressed as the mean ± standard errors (SEM). We used the one-way ANOVA, then post hoc Tukey's test to calculate P-values for continuous variables. All statistical analyses were performed using Origin (OriginLab Corporation, Northampton, Massachusetts, USA), version 8. The threshold for statistical significance was set at P=0.05 based on a two-sided test.

Results
Electroacupuncture (EA) and rivastigmine significantly reversed 6-OHDA induced spatial and reversal learning dysfunction, but not motor function in a PDD mouse model.
We used a Morris water maze for behavioral tests. In the first four days, acquisition behavior training was performed. Escape latency in each group of mice decreased day-to-day ( Figure 1). On acquisition days 3 (Control = 4.40 ± 0.8 sec, PDD = 9.82 ± 1.52 sec, EA = 5.04 ± 0.58 sec, Riva = 4.75 ± 0.87 sec; P=0.001) and 4, PDD mice showed significantly longer escape latency than other treated mice, indicating that PDD mice displayed impaired spatial memory. This impairment was reversed by EA and oral rivastigmine. After acquisition, we put the hidden platform in an opposite area to test reversal learning. PDD mice showed prolonged escape latency over all four reversal days (R1-R4). On reversal day 3, the escape latencies were: Control = 2.86 ± 0.46 sec, PDD = 9.80 ± 1.83 sec, EA = 4.6 ± 0.82 sec, Riva = 4.6 ± 1.03 sec; P=0.001. Similarly, learning impairment was reversed by EA or oral rivastigmine ( Figure 1C). All mice exhibited similar swimming speed on seven of eight days. Only on reversal day 2 (R2), mice in the drug group showed faster swimming ( Figure 1D). PDD mice apparently did not suffer motor dysfunction as expressed by bradykinesia. Thus, prolonged escape latency was solely due to cognitive decline.
Inflammatory cytokines were increased in PDD mice plasma and further attenuated through EA or rivastigmine treatment.
The effect of EA and rivastigmine treatment on TRPV1 Behavior tests showed impairment in both spatial learning and cognitive flexibility in PDD mice. Associated changes in proteins in brain samples were assessed by Western blotting. We focused on the hippocampus for spatial learning and the PFC for reversal learning. In both areas, TRPV1 and downstream molecules (pPKA, pPI3K, pPKC, pAkt, pmTOR, pERK, and pCREB) were upregulated in PDD group mice. This increase in expression was reversed by EA and oral rivastigmine. Interestingly, EA treated mice showed an increase in α7 nicotinic receptors and parvalbumin level in these brain areas (Figures 3 and 4).
The effect of EA or rivastigmine treatment on TRPV1 expression in the hippocampus and PFC via immunofluorescence technique.
Western blotting analysis showed TRPV1 upregulation in the hippocampus and PFC. We used  immunofluorescence to stain TRPV1 positive cells in the hippocampus ( Figure 5) and PFC ( Figure 6). We observed consistent Western blotting results that showed an increase in TRPV1 expression in PDD mice. This increase could be reversed by EA or oral rivastigmine.

Discussion
A 2019 review article summarized recent studies of neuroinflammation in PD-associated neurodegeneration. Proinflammatory cytokines(IL-1β, IL-6, and TNF-α), mediated by the microglia and astrocytes play an important role in this process (32). Using 6-OHDA to induce neuroinflammation in a PDD mouse model, we increased plasma proinflammatory cytokine concentrations of IL-1β , IL-5, IL-6, and TNF-α. Consistent with a previous study, neuroinflammation paralleled TRPV1 activation in the hippocampus and PFC (8). The summary of our finding is shown in Figure 7.
Interestingly, out of the 18 studies we reviewed that focused on the modulation of TRPV1 via acupuncture (Table 2), 13 studies showed that acupuncture decreased TRPV1 expression to relieve symptoms, while the other five reported an increase in TRPV1 expression. This discrepancy may be due to the bidirectional modulations of both acupuncture (33) and neuroinflammation byTRPV1 (8). Since most animal studies on PD investigated motor   symptoms such as bradykinesia and rigidity, we focused on two cognitive domains: spatial memory and cognitive flexibility. Previous research used the same animal PDD model (30) and showed that electroacupuncture rescued learning and long-term potentiation deficits. Authors reported that electroacupuncture (EA) on the bilateral KI3 reduced neuronal excitotoxicity by regulating N-methyl-d-aspartate (NMDA) receptor functions. We used a similar method and analyzed TRPV1 and related signaling, along with a behavior test for reversal learning to investigate cognitive inflexibility in PDD mice.
Some patients with PD clinically display rigid thinking and have difficulty altering their ideas. Cools et al. focused on cognitive flexibility (34) and used a strict method to simplify concept formation, learning, working memory, and a general slowing of cognitive processes. They reported strong evidence of cognitive inflexibility in patients with PD, with disrupted interactions between the frontal cortex and striatum. Another recent study showed that the dysfunction of parvalbumin (PV)-positive GABAergic interneurons (PVIs) within the PFC was associated with cognitive inflexibility (35). Parvalbumin is a calcium-binding low molecular weight protein, typically 9-11 kDa (36). We examined parvalbumin in both the PFC and hippocampus. Interestingly, we found that parvalbumin levels increased in mice treated with electroacupuncture but not after oral administration of rivastigmine. This finding was consistent with reports that electroacupuncture alleviates anxiety-like behavior in adult mice (37). Another study investigated the disrupted balance between inhibition, such as parvalbumin-positive GABAergic interneurons, and excitation within the neuronal networks for acupuncture and epilepsy (38). That study showed that parvalbumin was more GABAergic, while TRPV1 activation was more glutamatergic (39). Because of this, we speculated that increased GABAergic effects of electroacupuncture reduced glutamatergic effects of TRPV1 and thus improved cognitive flexibility of mice.
Another difference between EA and oral rivastigmine is the effect on swimming speed. We found that mice administered with rivastigmine swam faster on all eight testing days. However, only results from reversal day 2 (R2) showed statistical significance (P=0.03). We   (40). This treatment improved gait stability and might reduce fall frequency. Other studies (41) found that patients with PD need to concentrate to compensate for impaired gait stability and that oral rivastigmine might improve gait by improving cognitive function and attention (42). Clinical studies showed that LR3 (Tai Chong) is the most common acupoint for PD treatment, other than GB34, GV20, EX-HN1, GB20, LI11, ST36, and KI3 (Tai Xi) (23). Using functional MRI to evaluate acupuncture effects in the brain, KI3 was shown to improve cognitive function in patients in human studies (43). Similarly, bilateral EA using KI3 showed positive effects in the hippocampus in a previous PDD mouse study (30). According to traditional Chinese medical history, although the pathological location of cognitive decline is in the brain, an essential factor lies in the kidney, hence, KI3 (Tai xi) is considered as a primary acupoint used clinically for treating cognitive disorders.

Conclusion
Our study has found that PDD involves neuroinflammation and that the modulation of TRPV1 and related signaling via treatment with EA and oral rivastigmine might alleviate this inflammation. Therefore, TRPV1 may be a target for the treatment of patients with PDD. Since the treatments used here affect different molecular pathways, further studies are needed to clarify their difference in detail.